Abstract of research The finding that most patients with virologic failure (HIV-1 RNA >=1,000cp/mL) on second-line PI- containing regimens lack PI-resistance mutations in the protease (PR) gene is one of the main enigmas of antiretroviral drug resistance research. We hypothesized that env and gag sequences from these patients contain compensatory mutations, specifically in the Env, that confer PI resistance. A role of the gp41 CT in PI resistance may be linked to the role of virus maturation, triggered by PR, in activating Env fusion activity. In recent years, increasing numbers of clinical reports have observed failure of DTG-containing therapy in the absence of integrase (IN) mutations, suggesting that mutations outside IN may confer resistance in these patients. Our studies demonstrated the first instance of de novo selection of Env mutations that confer resistance to Dolutegravir (DTG) in vitro (unpublished data). We attribute this phenotype to the ability of the Env mutants to mediate highly efficient cell-to-cell transmission, resulting in an increase in the multiplicity of infection. In addition, up to 20-, 6- and 24-fold reduction in susceptibility to ATV, DRV and LPV, respectively, was observed in absence of PR mutations among HIV-1 CRF_02AG and subtype G infected patients. These findings have broad implications for our understanding of Env and Gag functions and the evolution of HIV-1 drug resistance. Our goal is to identify factors that predict virological failure (VF) on ATV/r or LPV/r as patients with VF on a PI-containing regimens have suboptimal future treatment and may be at an increased risk for developing integrase resistance inhibitor on a third-line regimen. A collaborative team involving investigators led by the Institute of Human Virology Nigeria will exploit robust longitudinal cohorts (Cameroon, Nigeria and Uganda) of patients on 2L regimens, coupled to well- characterized and archived clinical specimens that underpin a rigorous nested case-control study design. To test these interlocking hypotheses, we propose to: (i) use our newly developed HIV-xGen next generation sequencing method to extend understanding of viral evolution and HIV drug resistance across the viral genome and to determine whether there is effect modification by non-B HIV-1 subtypes, (ii) characterize recombinant viruses carrying mutations identified in Aim 1, using established phenotypic assays to determine their effect on drug susceptibility and site-directed mutagenesis, and lastly (iii) determine differences in replicative fitness on drug susceptibility with use of a yeast recombination-based cloning system. Our plans to study these patients and their viruses (subtype A, C, D, G, and CRF02_AG, CRF43_02G, HIV-1 O and HIV-1 N) provides a unique opportunity to determine the phenotypic and clinical significance of genotypic changes in HIV-1 gag, gag-pol and env genes. All in all, this proposal will permit exciting epidemiological, clinical, and public health research opportunities.